The preclinical prediction of adverse reactions of a drug candidate is a difficult and elusive task. This is primarily attributed to the lack of scientific understanding of the mechanism of some adverse reactions and also to the lack of animal models. The prediction is especially difficult in the case of idiosyncratic drug reactions, which have very low frequency of occurrence, no apparent dose-response relationship, and no animal models for evaluation. (Uetrecht, J. P., Chem Res Toxicol, 1999, 12(5), 387–95; Park, B. K., et al, Toxicology, 2000, 153(1–3), 39–60; and Li, A. P., Chem Biol Interact, 2002, 142(1–2), 7–23). At present, there is not a commonly accepted experimental approach to predict idiosyncratic drug reactions.
Most of the drugs that are associated with idiosyncratic drug reactions form reactive metabolites that react with endogenous nucleophiles, including proteins (Li 2002). Two hypotheses have been proposed that link reactive metabolites with idiosyncratic reactions, namely the hapten hypothesis and the danger hypothesis, both involve the triggering of immune response following the insult from reactive metabolites. (Uetrecht 1999; Park, Kitteringham et al. 2000; and Park, B. K., Naisbitt, D. J. et al., Toxicology, 2001, 158(1–2), 11–23). In either case, it seems logical to screen for reactive metabolites preclinically to reduce the risk of such drug reactions and to decrease the attrition rate of new drug candidates.
Thiol-containing nucleophiles have long been used for the trapping of reactive intermediates. Glutathione is the most important physiological thiol containing nucleophile. Glutathione is a tripeptide γ-glu-cys-gly produced endogenously. It is an important cellular component that is crucial for the cellular homeostasis of redox potential and a natural defense against oxidative stress. Glutathione is also regarded primarily as a detoxification agent because it reacts with many known reactive metabolites and the resulting glutathione adducts are usually nontoxic and excreted readily from the human body. Because of its ability to react with a variety of reactive metabolites, glutathione has been widely used as a in vitro trapping agent for the characterization and mechanistic study of reactive metabolites. There are several approaches that have been reported for the preclinical detection and screening for glutathione adduct formations, namely, neutral loss LC-MS/MS screening and tritiated glutathione trapping. Neutral loss LC-MS/MS screening utilizes the characteristic fragmentation pattern of glutathione and glutathione adducts when they are subjected to the collision induced decomposition (CID) in the second quadrapole of a triple quadrapole mass spectrometer (Chen, W. G. et al., Adv Exp Med Biol, 2001, 500, 521–4). This method is rapid and relatively sensitive, however it is not quantitative. The other method utilizes radiolabeled glutathione (tritiated) and adducts can be quantitatively determined by radioactivity counting. Technical and financial concerns have limited the use of the radiolabeled glutathione method. Adequate separation of the glutathione adducts from the unreacted material is challenging and results in insufficient sensitivity. Further, the use of radioactivity requires special facilities and creates environmentally hazardous waste material. Finally, the radiolabeled glutathione method is relatively expensive.
Thus, there exists a need for a sensitive, quantitative, and cost-effective method to trap reactive metabolites in vitro. The present application answers this need.